Variations in the nucleotide sequence of DNA impact if and how an organism develops diseases, and respond to pathogens, chemicals, drugs, vaccines and other agents.
Single-nucleotide variations, such as single-nucleotide polymorphisms (SNPs) or point mutations, play an important role in many human diseases. As genetic markers, SNPs can be used to trace generational inheritance patterns associated with specific diseases. As diagnostic markers, point mutations can be used for early cancer detection. Current methods for single nucleotide variation detection (e.g., genotyping of known SNPs) typically require enzymatic reactions such as primer extension, ligation, and cleavage, making these methods time consuming and expensive. Hybridization-based methods that rely on optical, electrical, or electrochemical signals for discrimination readout are considerably simpler in practice. However, most of these methods rely on differences in the free energy of probe/target binding for SNP differentiation (i.e., hybridization probes bind preferably to the fully matched target rather than the single-base mismatched targets, such as molecular beacons). Such differences in binding free energy are often small and can vary significantly on the basis of target sequence. Therefore, sophisticated probe design algorithms and use of hybridization enhancing moieties are often necessary. Further, optimized assay conditions (such as elevated temperature for molecular beacon discrimination) are often required, which also limit their use at point-of-care settings.
Therefore, there continues to be a need for rapid and precise screening of small genetic variations such as SNPs. The present disclosure meets such needs by removing or minimizing the disadvantages of existing methods, and further reducing costs associated with such probes and methods.